利用荧光探针直接测定线粒体活性氧的形成

目的与方法：根据荧光探针－还原型二氯荧光素（2‘,7‘-dichlorodihydrofluorescin,DCFH)可与活性氧（reactive oxygen species,ROS)反应生成荧光物一氧化型二氯荧光素（2‘,7‘-dichlorofluorecin,DCF)的原理,设计了利用荧光分光光度计直接定量检测线粒体活性氧生成并可观察在各种实验条件下线粒体活性氧产生动态变化的方法。结果与结论：线粒体在态4呼吸状态下,DCF的荧光强度随时间呈线性增加,表明活性氧以恒定速率产生。将荧光强度随时间变化的数据点拟合,线性回归直线斜率与活性氧产生的速率呈正比,测定中加入叠氮钠和丙二酸可分别使线粒体活性氧产生增加和减少。DCF荧光强度增加速率与线粒体浓度在一定范围内呈线性关系。复管实验表明重复性良好。

Determination of reactive oxygen species in mitochondria by fluorometric probe

AIM AND METHODS: Based on the reaction that 2',7'-dichlorodihydrofluorescein (DCFH) can be oxidized by reactive oxygen species (ROS) to yield the highly fluorescent 2',7'-dichlorofluorescin (DCF), ROS production in mitochondria can be observed dynamically as well as quantified directly by spectrofluorometer. RESULTS AND CONCLUSION: DCF fluorescence showed linear increase in State 4 mitochondria, which suggest ROS produced at constant rate. The slopes of the linear increase in fluorescence with time were computed performing a linear regression that took into account all relevant data points in selected time windows. The slopes were proportional to ROS production in mitochondria. Addition of sodium azide and malonic acid increased and decreased the rate of ROS production respectively during measurement. DCF fluorescence varied linearly with increasing concentration of mitochondria, which showed quantitative relations in definite range. Repeated measures showed low coefficients of variation. This method is reliable and efficient for determining ROS in mitochondria.